PROJECT SUMMARY Inability to generate self-renewing hematopoietic stem cells from pluripotent stem cells (PSC) has proven to be a major bottleneck that inhibits the use of in vitro engineered hematopoietic cells for therapeutic purposes. We hypothesize that defective induction of the correct transcriptional networks governing HSC self-renewal during the critical stages of hematopoietic development prevents the emergence of fully functional HSC in an in vitro setting. Guided by the transcriptional profile of highly purified human fetal liver HSC, we identified key transcriptional regulators that govern self-renewal in developing human HSC with the goal to use this knowledge to develop new strategies to improve the function of in vitro derived hematopoietic cells. We identified MLLT3/AF9, a component of superelongation complex (SEC) as a novel regulator of HSC stemness. MLLT3 was highly expressed in the GPI80 FL-HSPC population that is highly enriched for the true self- renewing HSC during human development, and downregulated during differentiation and in vitro culture. Lentiviral knockdown of MLLT3 in human fetal liver and cord blood HSC showed its importance for protecting HSC function in vitro and in vivo, while overexpression of MLLT3 improved the expansion of fetal liver and cord blood HSPCs and hESC-derived HSPCs in culture. We will now identify MLLT3 target genes and test the hypothesis that MLLT3 facilitates efficient elongation of HSC stemness genes (Aim 1). We will then examine whether MLLT3 overexpression using constitutive or transient expression systems increases the in vivo engraftment ability of human HSCs and hESC-derived HSPCs (Aim 2). Finally, we will investigate mechanisms that regulate MLLT3 expression in human HSCs, focusing on HSC specific regulatory elements and testing the importance of a poorly characterized shorter isoform that is highly expressed in HSCs (Aim 3). Understanding how MLLT3 functions as a key upstream regulator that can confer ?stemness? in developing HSCs will both increase our knowledge of the fundamental regulatory mechanisms governing human HSC as well as pave the way for developing novel approaches for the in vitro generation of HSC for therapeutic use.